Genistein, an isoflavone, is a member of the dietary phytoestrogens that are believed to have chemopreventive effect and is currently undergoing clinical trial for prostate cancer prevention. Genistein is widely consumed by general public in the form of soy isoflavone supplements. Despite its proven in vitro anticancer activity, genistein and other isoflavones have poor bioavailability. In addition, whether the parent compound or one of its metabolites is the active species has not been carefully determined. This is a serious concern since the in vivo concentration of genistein achieved (in nM range) after soy food consumption or administration of soy isoflavone supplements are far lower than those necessary to show anticancer activity in vitro (in muM range). Therefore, the long-term goal of our study is to increase our understanding of the intestinal disposition of isoflavonoids (and other phytoestrogens) so that we can improve the bioavailability of active species and ensure their safe and effective use as chemopreventive agents. In the present proposal, we will focus our efforts on the study of absorption/excretion pathways of isoflavones and their conjugates in the intestine via the use of in vitro, in situ and in vivo models, and determine how intestinal disposition affects the biotransformation and bioavailability of isoflavonoids in vivo. The aims of this study are abbreviated as: (1) to determine region-dependent (i.e., duodenum, jejunum, ileum, and colon) hydrolysis of glycosides; (2) to determine the role played by glucose transporters in the transepithelial transport of glycosides; (3) to define the biological basis of glycoside efflux in the intestine; (4) to identify the UGT isoform(s) responsible for the intestinal metabolism of isoflavones and to determine the efflux mechanisms responsible for intestinal secretion of UGT conjugates; and (5) to determine how intestinal disposition processes will affect the in vivo biotransformation and bioavailability of genistein and its analogs. These proposed studies will provide an explanation as to which mechanisms are responsible for the poor bioavailability of this class of phytoestrogens. They will form the basis for future efficacy studies to determine if improved bioavailability (through manipulating intestinal absorption and biotransformation) will translate into increased efficacy. Such efficacy studies could help to identify the active species responsible for observed anticancer activities, important for the understanding of the mechanisms of action of isoflavones.